The alpha-factor receptor of yeast (encoded by the STE2 gene) mediates control of the cell-cycle and transcription. It is member of a large family of integral-membrane, G protein-coupled receptors. Others include an angiotensin receptor (encoded by the mas proto-oncogene), the Beta- adrenergic receptor, and rhodopsin. I propose to study three aspects of receptor function: 1. The alpha-factor binding site. 2. The coupling of receptor activation to activation of G protein and to specific cellular responses. 3. The role and mechanism of alpha-factor induced internalization of receptor. In one approach to these problems, components of the signal transduction system of S. kluyveri will be introduced into S. cerevisiae. These yeasts are divergent enough that interactions between ligand and receptor (for example) exhibit substantial species specificity, though they are similar enough that S. kluyveri STE2 functions fairly well in S. cerevisiae. Hybrid receptors will be used to characterize ligand specificity determinants. To extend the analysis to interaction of receptor with G protein, S. kluyveri G protein subunit genes will be cloned. Another approach will be the identification of mutations of STE2 that affect specific functions. Mutations that alter ligand specificity will be used to help map the alpha-factor binding site. Mutations that affect the ability of receptor to activate some, but not all, responses will be used to study the requirements for receptor signaling. Mutations that affect only receptor internalization, will be used to study the role of internalization in receptor function. The goal of this research is to understand how the alpha-factor receptor works and to identify regions of the receptor involved in specific functions.